WO1999037750A1

WO1999037750A1 - Method and device for cell lysis

Info

Publication number: WO1999037750A1
Application number: PCT/FR1999/000105
Authority: WO
Inventors: Michel Chevalier
Original assignee: Aventis Pasteur
Priority date: 1998-01-21
Filing date: 1999-01-20
Publication date: 1999-07-29
Also published as: JP2002500878A; NZ505865A; FR2773818B1; AU2060499A; FR2773818A1; EP1049766A1; CA2319021A1; AU756179B2

Abstract

The invention concerns a method and a device for cell lysis which consists in continuously producing a liquid mixture of bacteria or eukaryiotic cells and a lysis agent, and in immediately causing said mixture to flow in a constant flux inside a pipe (7), the flow rate of said flux being adapted, according to the pipe (7) length and diameter, so as to obtain a substantially homogeneous lysate at said pipe (7) outlet.

Description

1

Cell lysis method and device

The present invention relates to a method and a device for lysis of bacteria or eukaryotic cells, as well as extraction and purification of nucleic acids, and in particular plasmids, from bacteria or eukaryotic cells containing these plasmids.

The production of plasmids of interest, and in particular of plasmids into which a gene or a coding DNA sequence has been inserted, is carried out by multicopy of these plasmids in bacteria capable of producing a large number of these plasmids, and in particular in certain strains of Escherichia coli highly productive of plasmids and often already used in the laboratory or on an industrial scale.

One of the applications which requires significant production of a plasmid, on an industrial scale, is the manufacture of medicaments or vectors of prophylactic or therapeutic interest based on a naked plasmid or associated with means of penetration into the recipient host cells. Such applications are described for example in patent application WO 90/11092.

There are several techniques of bacterial lysis allowing the extraction of the plasmids, followed by a separation, and therefore of a purification of these plasmids. The most commonly used technique is the alkaline lysis technique which uses an alkaline lysis agent such as a sodium hydroxide + SDS preparation (sodium dodecyl sulfate), followed by neutralization with an acid agent, such as potassium acetate. This neutralizing agent also has the effect of precipitating all of the bacterial constituents, including genomic DNA, the supernatant essentially containing plasmid DNA. The supernatant can then be separated from the precipitate by centrifugation or filtration (Birnboim Methods in Enzymology (1983) 100: 243). The alkaline lysis technique is particularly recommended for lysing bacteria; but it can just as easily be used to lyse eukaryotic cells.

This technique, which is suitable for the laboratory scale, is painful to implement on an industrial scale because the lysate resulting from the action of the alkaline agent on the bacteria constitutes a very viscous suspension, 2 so that the lysate being formed must be homogenized by agitation. Likewise, after neutralization with acid, homogenization by stirring is necessary.

This stirring is delicate and it is generally carried out manually, the operator gently shaking the bottles or flasks containing the lysis medium. Insufficient agitation provides poor quality lysis while excessive agitation tends to fragment genomic DNA, which subsequently mixes with the plasmids. In both cases, a decrease in the yield of plasmid DNA is observed. Consequently, the operator's hand is an essential guarantee of the success of the operation.

In order to automate the lysis process, it has already been proposed, in document WO 97/23601, to pass, continuously, the suspension of cells to be lysed and a solution of a lysis agent through a static mixer. 'a sufficient length to complete the lysis. The resulting lysate can then be brought, by a pipe, to a second static mixer into which also enters a solution of a precipitating agent.

Such a method therefore implements a specific means, namely a static mixer, which replaces manual agitation of large volumes with continuous agitation over the entire length of the mixer. The use of such a mixer, in addition to requiring the purchase, maintenance and cleaning of the device, imposes a fixed period of contact with the lysis agent, with no practical possibility of controlling it or modulate it. In addition, the stirring maintained in the mixer, even if it is preferable to the poorly controlled stirring of discontinuous volumes, can cause breaks in, or degradations of cellular constituents and in particular of nucleic acids.

Another method, described for example in document WO 96/36106, also makes it possible to establish continuous lysis, but this time without using agitation means. This process consists of (i) preparing a mixture of a bacterial suspension and an agent, such as lysozyme, allowing this mixture to incubate for approximately one hour in order to weaken the bacterial wall, then (ii) passing a flow of this mixture inside a pipe heated to high temperature (70-100 ° C). The action of heat promotes lysis. The disadvantage of such a solution is that it requires heating means to 3 high temperatures and an adaptation of the temperature to the different special cases that may be encountered.

The present invention proposes to remedy these drawbacks and to provide a cell lysis process, applicable in particular to the extraction and purification of nucleic acids such as plasmids from bacteria or eukaryotic cells, which is capable of '' be implemented without manual intervention and under extremely economical conditions.

Another objective of the invention is to provide a method which makes it possible to control in an extremely precise manner the conditions and the duration of the lysis and of the extraction and this for all the cells present.

Another objective is to provide a method making it possible to establish substantially homogeneous lysis conditions for a population of cells.

Another objective is to provide a process capable of being implemented in a closed environment, free from contamination, which is an advantage for the pharmaceutical quality of the products sought, for example plasmids.

Another objective is to obtain a reduction in the duration of the cell / lysis agent contact, which is necessary to complete the lysis.

Another objective is to provide a device for implementing this method, a simple and inexpensive device.

Another objective is to provide on an industrial scale, plasmid preparations with a high yield and appreciably improved compared to that of the preparations which can be obtained according to the methods of the prior art. The invention is based on the unexpected discovery that it is possible, by respecting a certain number of parameters, to ensure a homogeneous and controlled lysis of cells, and in particular of bacteria, by simple continuous mixing, in a usual pipe, of a suspension of cells with a lysis agent, despite the expected high viscosity, without using any of the means of the prior art such as a static mixer or high temperatures.

The invention therefore relates to a cell lysis process in which a liquid mixture of cells and a lysis agent is produced continuously, and 4 immediately causes this mixture to flow under a constant flow, inside a pipeline, the flow rate of this flow being adapted, as a function of the diameter and the length of the pipeline, so as to obtain a cell lysate substantially homogeneous with the outlet of said pipe. Obtaining a homogeneous lysate results in a sharp drop in turbidity and the appearance of a mixture transparent to the eye.

Preferably, the pipeline has a small internal diameter so that the mixture forms almost instantly in a homogeneous manner, without the formation of separate liquid veins. This diameter can be determined experimentally. In general, a diameter of the order of 1 cm or preferably less meets this definition, and a diameter of between 2 and 8 mm is preferred.

It should obviously be understood that if the diameter and the flow rate are elements of the process fixed in advance, it is just as possible to adapt the length of the pipe so as to obtain the desired effect. For example, for a given diameter and flow rate, it is enough to play on the length of the pipe, e.g., using a simple flexible pipe that is cut to the desired length.

The minimum length of the pipeline from the meeting point of the cell suspension and the lysis agent, which is necessary to travel to reach the lysis state, can be easily determined by simple observation through a pipeline transparent, from the reduction in the turbidity of the mixture until the appearance of a lysate transparent to the eye. A pipe length of the order of 10 cm to a few meters is generally suitable.

One of the advantages of the invention consists in the homogeneity of the mixture during lysis (the duration of lysis is substantially identical for all bacteria); which ultimately allows a homogeneous lysate to be obtained. This is achieved by the appropriate choice of the parameters of the invention (diameter, and length of pipe, flow rate), without having to use stirring or heating means.

Preferably, the mixture of cells and of lysis agent is produced by introducing, into the aforesaid pipeline, a flow of cells, for example of a 5 cell suspension, and a flow of a lysis agent solution, so that the flow of the flow of this mixture produces rapid homogenization, which is almost instantaneous, if using a reduced diameter pipeline.

The lysis agent can be a chemical agent, for example an alkaline agent, such as a sodium hydroxide solution + SDS, preferably a 0.2 M NaOH / 1% SDS mixture. It can also be a hypotonic solution compared to the cellular environment, intended to cause osmotic shock. In the case where the bacteria are simply transferred to a hypotonic solution, they have been treated beforehand so that their wall is weakened, by an agent such as lysozyme. Treatment with an alkaline agent is particularly suitable for bacterial lysis, while treatment with a hypotonic solution is particularly suitable for lysis of eukaryotic cells.

In the case where an alkaline lysis agent has been used, it is preferable to add to the lysate, a neutralizing agent. Indeed, the addition of this agent makes it possible to stop the degrading action linked to the alkaline agent, once a complete and homogeneous lysis has been obtained. In the latter case, another advantage of the invention lies in exercising control over the period during which the cells are subjected to strongly alkaline pH conditions. This makes it possible to very easily implement the optimal duration conditions resulting in a total and homogeneous lysis while avoiding prolonging the action of the alkaline agent beyond the time necessary and sufficient to complete the lysis in order to avoid any action. deleterious in particular on DNA. The neutralizing agent which is added to the lysate arriving at the end of the pipeline may preferably be sodium or potassium acetate, for example 3 M potassium acetate. It is advantageously chosen to so as to obtain a final pH close to 5.5 thanks to the addition of HCI 12N.

On the other hand, if the aim pursued in fine consists eg in extracting plasmids, it is desirable to separate these plasmids from the rest of the cellular constituents. This separation is carried out by precipitating these cellular constituents, including genomic DNA, by adding a precipitating agent to the lysate; the plasmids then remaining in the supernatant. As the precipitating agent, sodium or potassium acetate can be used, for example 6

example of potassium acetate 3 M. It is advantageously chosen so as to obtain a final pH close to 5.5 thanks to the addition of HCl 12N.

A precipitation agent such as sodium or potassium acetate also serves as a neutralizing agent, when the alkaline lysis technique is used.

The subject of the invention is also a method of extraction and / or purification of nucleic acids, in particular of plasmids, from a cell suspension, in which (i) the lysis method according to the invention in order to obtain a cell lysate and, continuously with the lysis process, (i) the cell lysate is treated with a precipitating and / or neutralizing agent, in order to obtain a preparation comprising a supernatant containing the plasmid DNA and a precipitated or flocculated phase containing the majority of cellular elements including genomic DNA. To do this, advantageously, (i) a continuous mixture of a cell suspension and a liquid preparation (solution) of a lysis agent is produced, at a first determined meeting point, starting from from which a constant flow of said mixture is established in a pipe, in order to homogenize the mixture of the suspension and of the lysis agent, (ii) this mixture is maintained in this constant flow for a determined duration, and at the end of this duration (iii) adding, at a second determined meeting point, a solution of a precipitating agent; said duration being determined by the distance separating said first and second meeting points and by the speed of movement of the mixture (or linear flow) over this distance. Once the precipitation has been carried out, the plasmids remaining in the supernatant are separated in any way from the rest of the precipitated or flocculated cellular elements.

This plasmid extraction process can advantageously be implemented in the following way: (i) a flow of the cell suspension is established in a first channel;

(ii) establishing a flow of the lysis agent in solution in a second pipe which flows into the first pipe (or vice-versa) at a first meeting point to form a third pipe; 7

(iii) establishing a flow of a precipitating agent in a fourth pipe which flows into the third pipe (or vice-versa) at a second meeting point located downstream of the first meeting point, to form a fifth pipe; (iv) a flow of the cell / lysis agent mixture produced at the first meeting point, in the third pipe (between the first and the second meeting point) is established at a linear flow rate adapted as a function of the diameter and the length the third pipe, so as to allow the homogenization of the mixture and the obtaining of a substantially homogeneous cell lysate at the second meeting point; and

(v) in the fifth pipe, a flow of the preparation obtained at the second meeting point is established, by mixing the cell lysate with the precipitation agent; and (vi) a preparation comprising a supernatant containing the plasmid DNA and a precipitated or flocculated phase containing the majority of the cellular elements including the genomic DNA is recovered at the outlet of the fifth channel.

Thereafter, it is also possible to proceed, in any manner, with the separation of the supernatant from the precipitated or flocculated phase, preferably continuously from step (vi) of recovery.

The following table indicates, by way of example, various experimental conditions (length of pipe, diameter, flow rate, duration of contact with the alkaline lysis agent) which make it possible to obtain a complete and homogeneous bacterial lysis, by the technique of alkaline lysis. The bacterial and lysis agent concentrations remain identical from one test to another. Length of Diameter Flow rate Contact time Pipe with lysis agent

28 cm 0.3 cm 160 ml / min 2 seconds

26 cm 0.7 cm 160 ml / min 15 seconds

85 cm + 0.7 cm 160 ml / min 4 minutes 500 ml tank

The time required to obtain a complete and homogeneous lysis, taking into account the usual proportions of suspension and of lysis agent, can be very substantially reduced and even less than one or more minutes, for example 5 min and even reduced to such low values. one or two seconds, contrasting with the ten minutes required in the prior art.

It is also understood that, by virtue of the invention, it provides completely homogeneous lysis, precipitation and / or neutralization conditions over time, over the entire cell suspension which is poured through the pipeline.

The proportion of flow rates, and therefore of mixed volumes, preferably meets the following definitions: - bacterial suspension / lysis agent (for example sodium hydroxide mixture +

SDS): between 1/4 and 3/4, and preferably of the order of 1/2,

- alkaline lysis agent / neutralizing acid agent, preferably potassium acetate: between 1 and 2 and preferably of the order of 1, 3.

Preferably the concentration of the bacterial suspension is of the order of 170 grams (by wet weight of bacteria) / liter of a conventional buffer (for example Tris EDTA) + glucose.

Preferably, the cell suspension is stored at low temperature and directed at this same temperature in the pipe towards the meeting point with the lysis agent, this temperature preferably being of the order of 4 ° C. The lysis agent can be maintained and transported at ambient temperature and the precipitating and / or neutralizing agent is preferably maintained at low temperature, such as 4 ° C. 9

The invention also relates to a device for implementing this process, characterized in that it comprises, from a source of cell suspension, such as a reservoir, a pipe allowing the establishment of a flow of cell suspension, from a source of a lysis agent such as a reservoir, a second line allowing the establishment of a flow of the lysis agent, said first and second lines leading to a first meeting point into which they open into one another, - a third pipe of small diameter and of determined length extending from said first meeting point, and means, such as pumping means, for the establishment of flows in said pipes; the length, the diameter and the flow rate in said third pipe being adapted so as to obtain a substantially homogeneous mixture resulting in a substantially homogeneous lysate.

The device may also comprise: from a source of neutralizing and / or precipitating agent, such as a reservoir, a fourth pipe leading to a second meeting point at the end of said third pipe so that the pipes open into one another, said third pipe having a length determined by the distance between said first meeting point and said second meeting point; from said second meeting point, a fifth small diameter pipe leading to recovery and / or separation means; and and means, such as pumping means, for establishing the flows in said fourth and fifth pipes.

Preferably all the pipes have small diameters as defined above.

The small diameters of said pipes can be identical or different. The differences in diameter between pipes can be determined by the flow establishment means. 10

The means for establishing the flows can advantageously be pumps, preferably one or more peristaltic pumps making it possible to establish, in the various pipes, the flow rates, and therefore, taking into account the diameters of the pipes, the desired speeds. Advantageously, it is possible to use, for the establishment of two or more of the flows, the same pump, for example, a peristaltic pump with several parallel channels, so as to ensure, even in the event of untimely variation of the pump flow rate , a constant proportionality between said flows. Of course, if the quantities to be treated are particularly large, the arrangement of the pipes according to the invention can be split, tripled or multiplied, preferably with flow establishment means, such as a pump, single or more pumps joined together so as to maintain, in all circumstances, a constant proportionality of the flows in each of the installations.

The outlet from one pipe to the other at a meeting point can be made at any angle. Generally it is preferred that one of the pipes opens substantially perpendicularly into the other but it is also possible to tilt the axes of the outlets.

Preferably, no particular means of homogenization is provided such as a baffle or obstacle in the pipeline at the meeting points; nor heating means allowing to reach high temperatures (higher than 60 ° C). Advantageously, the device according to the invention may include means for establishing and controlling temperatures so as to maintain the sources at the required temperatures. In particular for the cell suspension, the installation can be arranged so that the low temperature is maintained not only in the source but also in the first pipeline to the point where lysis begins.

An installation according to the invention makes it possible, for example, to treat a volume of bacterial suspension of the order of 1 to 5 liters per hour. 1 1

The mixture obtained downstream of the first meeting point exhibits great homogeneity throughout the duration of the treatment, as does the neutralized mixture downstream of the second meeting point, so that the separation means making it possible to separate, on the one hand , the plasmids remaining in solution, and, on the other hand, the other precipitated or flocculated cellular elements, work under constant conditions and contribute to the excellent reproducibility of the final purified plasmid preparation.

Furthermore, the final yield is significantly improved, which can exceed 50 mg of plasmid per 100 g of bacteria. Other advantages and characteristics of the invention will appear on reading the following description, given by way of nonlimiting example and referring to the appended drawing in which the single figure represents a schematic view of an implementation device of the method according to the invention.

The bacterial suspension to be lysed is a suspension originating from a culture of an E. coli strain in which a plasmid such as the plasmid pUC18 in Tris EDTA buffer has been multiplied with a bacterial concentration of the order of 200 g (weight wet) per liter.

The sodium hydroxide-SDS mixture is a 0.2 M NaOH / 1% SDS mixture.

The potassium acetate solution used as a neutralizing agent is 3 M; pH 5.5.

The device shown in the figure comprises a first container 1 containing the bacterial suspension. To this container are associated means for maintaining a temperature of the order of + 4 ° C. (not shown) and stirring means 2 making it possible to maintain the homogeneity of the suspension. From the source 1 extends a flexible silicone pipe having an internal diameter of 2.06 mm and forming the first section of pipe 3.

The sodium hydroxide-SDS mixture is contained in a reservoir 4 from which extends a second pipe, 5 with an internal diameter of 3.17 mm and made of the same flexible material. At the location 6 where the first meeting point is located, the pipe 3 opens perpendicularly into the pipe 5 so that at this location a mixture forms, causing rapid lysis of the bacteria. From point 6 extends a third section of 12 pipe, 7 to point 8 forming the second meeting point, the length of pipe 7 being 0.8 meters. This pipe 7 has an internal diameter of 7 mm (modular).

The potassium acetate solution is contained in a reservoir 9 from which extends a fourth pipe 10 also made of flexible material having an internal diameter of 2.79 mm opening into the pipe 7 at the meeting point 8, also perpendicular to line 7.

The container 9 is also associated with means for maintaining at low temperature + 4 ° C. From the second meeting point 8 extends a fifth pipe 11, formed subsequently by the pipe 7, this pipe 11 leading to a recovery tank 12.

It is understood that the diameters of the three pipes 3, 5 and 10 are in ratios such that the proportions of the internal sections of the pipes ensure, for the same speed of circulation of the liquids, the desired mixing proportions.

Consequently, the flexible pipes 3, 5 and 10 can pass through a single peristaltic pump 13, the rotary part 14 of which ensures the establishment of flows in the above proportions in the three pipes 3, 5 and 10.

The establishment of the flow rates in the desired proportions in the pipes 3, 5 and 10 of course determines the value of the flow rates downstream in the pipes 7, and 11.

The flow rates thus obtained are respectively 160 ml / min in 7 and 244 ml / min in 11.

The bacteria are subjected to the action of the sodium hydroxide + SDS mixture throughout the duration of the path of the liquid in the pipe 7, between points 6 and 8. This duration, in the example chosen, is 15 sec.

Finally, in the recovery container 12, a lysed preparation is obtained comprising two phases, namely a clear soluble phase containing the plasmids, substantially free of cellular element and an insoluble upper phase containing substantially the cellular elements. 13

The separation of these two phases is then carried out by conventional filtration or centrifugation techniques and the purification of the plasmids can still be continued according to the usual methods.

By way of verification, a comparative electrophoresis was carried out of the plasmid solutions obtained after alkaline lysis, either in continuous flow (as described in the example above) or by manual stirring for 10 min (the amounts of bacteria used in the two cases were similar). The revelation of the electrophoresis gel highlights the superiority of the continuous process in that the plasmid content is increased and that of the impurities lower.

Claims

14 CLAIMS

1. Cell lysis method according to which a liquid mixture of cells and a lysis agent is produced continuously, and this mixture is immediately made to flow under constant flow inside a pipe, the flow rate this flow being adapted, depending on the diameter and the length of the pipe, so as to obtain a substantially homogeneous cell lysate at the outlet of said pipe.

2. Method according to claim 1, wherein the pipeline has a reduced diameter facilitating very rapid homogenization of the mixture.

3. Method according to one of claims 1 and 2, wherein said small diameter is of the order of 1 cm or less.

4. The method of claim 3, wherein said small diameter is 2 to 8 mm.

5. Method according to one of claims 1 to 4, wherein the mixture of cells and lysis agent is carried out by introducing, into the said pipe, a flow of a suspension of cells and a flow of a solution lysis agent.

6. Method according to one of claims 1 to 5, wherein the lysis agent is of alkaline nature.

7. Method according to claim 6, characterized in that the lysis agent is a sodium hydroxide / SDS mixture.

8. The method of claim 6 or 7, wherein a neutralizing agent is added to the cell lysate arriving at the end of the pipeline. 15

9. The method of claim 8, wherein the neutralizing agent is sodium or potassium acetate.

10. Method according to one of claims 1 to 5, wherein the lysis agent is a hypotonic solution.

11. Method for extracting plasmid DNA from cells containing said DNA, in which (i) a lysis method according to one of claims 1 to 7 and 10 is used in order to obtain a cell lysate, and continuously the lysis process, (ii) the cell lysate is treated with a precipitating and / or neutralizing agent, in order to obtain a preparation comprising a supernatant containing the plasmid DNA and a precipitated or flocculated phase containing the majority of the elements cells, including genomic DNA.

12. Extraction method according to claim 1 1, in which:

(i) a flow of the cell suspension is established in a first channel;

(ii) a flow of a lysis agent is established in a second pipe which flows into the first pipe (or vice versa) at a first meeting point to form a third pipe;

(iii) a flow of a precipitating agent is established in a fourth pipe which flows into the third pipe (or vice versa) at a second meeting point located downstream of the first meeting point, to form a fifth pipe;

(iv) a flow of the cell / lysis agent mixture produced at the first meeting point, in the third pipe (between the first and the second meeting point) is established at a flow rate adapted as a function of the diameter and the length of the third channeling, so as to allow the homogenization of the mixture and the obtaining of a substantially homogeneous cell lysate at the second meeting point; and 16

(v) in the fifth channel, a flow of the preparation obtained at the second meeting point is established, by mixing the cell lysate with the precipitating agent; and (vi) a preparation comprising a supernatant containing the plasmid DNA and a precipitated or flocculated phase containing the majority of the cellular elements, including the genomic DNA, is recovered at the outlet of the fifth channel.

13. The method of claim 12, wherein the length of the pipe between the first and second meeting point is of the order of 10 cm to a few meters.

14. The method of claim 11, 12 or 13, wherein the precipitating and / or neutralizing agent is sodium or potassium acetate.

15. Method according to one of claims 11 to 14, in which (vii) the supernatant is further separated from the precipitated or flocculated phase, continuously from step (vi) of recovery.

16. Method according to one of claims 1 to 15, wherein the cells are bacteria.

17. Method according to one of claims 1 to 15, wherein the cells are eukaryotic cells.

18. Device for implementing the method according to one of claims 1 to 17, characterized in that it comprises, from a source of cell suspension, such as a reservoir, a pipe allowing the establishment a flow of cell suspension, from a source of a lysis agent such as a reservoir, a second pipe allowing the establishment of a flow of the lysis agent, said first and second pipes ending at 17 a first meeting point into which they open into one another, a third pipe of small diameter and determined length extending from said first meeting point, and means for establishing the flows in said first, second and third pipelines; the length, the diameter and the flow rate in said third pipe being adapted so as to obtain a substantially homogeneous mixture resulting in a substantially homogeneous lysate.

19. Device according to claim 18, characterized in that it further comprises: from a source of precipitating agent, such as a reservoir, a fourth pipe leading to a second meeting point at the end of said third line so that the lines open into one another, said third line having a length determined by the distance between said first meeting point and said second meeting point, from said second meeting point, a fifth small diameter pipe leading to recovery and / or separation means, and and means for establishing the flows in said fourth and fifth pipes.

20. Device according to claim 18 or 19, in which the means for establishing the flows are pumping means.